Aromatic hydrocarbons are toxic to most forms of life. In addition, certain compounds cause cancer in experimental animals. These effects are now thought to be due to electrophilic arene oxide intermediates that are formed by the enzymatic incorporation of one atom of molecular oxygen into the aromatic nucleus. Bacteria incorporate both atoms of molecular oxygen into the aromatic nucleus by a unique mechanism that does not involve the formation of free electrophilic intermediates. It is the main objective of this project to elucidate the mechanism of action of the enzyme systems from two different strains of Pseudomonas that are repsonsible for the oxidation of toluene and naphthalene to (+)-1(S),2(R)-dihydroxy-3-methyl-3,5-cyclohexadiene and (+)-1(R),2(S)-dihydroxy-1,2-dihydronaphthalene, respectively. Toluene dioxygenase consists of three protein components that have been purified to homogeneity. It is proposed that the electron transfer sequence in this system is as follows: NADH greater than ferredoxinTOL reductase greater than ferredoxinTOL greater than ISPTOL greater than greater than greater than cis-toluene-diol. The transfer of electrons from NADH to ISPTOL will be measured by spectrophotometric and electron paramagnetic resonance techniques. Further information will be obtained by measuring the redox properties of the components at controlled potentials. The iron-sulfur clusters in ferredoxinTOL and ISPTOL will be identified by extrusion experiments with appropriate thiol reagents. ISPTOL will be characterized with respect to its subunit composition and iron content. The role of the different subunits in the binding of iron, toluene and the oxygen fixation reaction will be determined by spectrophotometric, EPR and coulometric techniques. The structures of ferredoxinTOL and ISPTOL will be determined by X-ray diffraction studies. Three proteins (A, B and C) have been purified and shown to be essential for naphthalene dioxygenase activity. They will be characterized with respect to molecular weight, subunit composition, redox groups, redox potential, amino acid composition and EPR properties. The role of each component in naphthalene oxidation will be determined by spectrophotometric and EPR studies.